Expression of heterologous proteins in microbial production organisms can be a challenging process that often requires significant optimization due to misfolding of the proteins. Heterologous proteins are required for a number of processes, including for example metabolic pathway engineering, production of proteins for structure determination as well as biocatalytic processes. In metabolic pathway engineering for production of biochemicals, it is often required to functionally express a larger number of different enzymes. If one individual enzyme of the metabolic pathway is not correctly expressed or folded, the entire process will typically not be working optimally. There is also a very large market for heterologous proteins and peptides produced from microorganisms. Such peptides dependent on correct folding either directly in the production organism or during the subsequent post processing steps. Optimizing the translation, folding and stability of such target proteins is therefore of significant importance.
Although proteins can typically fold by themselves, most organisms have evolved mechanisms for controlling and aiding the process. Molecular chaperones typically assist in protein folding, and they can prevent polypeptide chains from aggregating before the correct protein folding has been achieved. Chaperones can either actively participate in protein folding using an energy dependent mechanism, or they can passively bind peptide chains, thereby preventing unwanted protein aggregation (Rayees et al 2014). Most molecular chaperones fall into a few conserved protein families, including Hsp100s (CIpB), Hsp90s (HtpG), Hsp70/Hsp110 (DnaK), Hsp60/CCTs (GroEL), as well as small heat shock proteins (IbpA/B). The chaperones bind to hydrophobic residues that are abnormally exposed to the cytosolic environment, and are thus prone to associate and form stable inactive aggregates. Chaperones are typically induced during stress conditions, and the proteins are often referred to heat shock proteins (Hsp). Expression of chaperones may differ from organism to organism, and this may contribute to the lack of predictability of folding of heterologously expressed proteins.
Several strategies for improving folding and expression of heterologous proteins are known. These include the use of protein expression and solubility tags, which are either short peptide or protein tags fused to the N-terminus of proteins. Those tags are supposed to function as folding scaffolds thereby helping to increase translation and folding of proteins with poor folding properties (Marblestone et al. 2006). Another strategy for improving protein folding includes the truncation of unstructured hydrophobic parts of the protein (Dyson et al. 2004).
A more efficient way to optimize protein expression would be to screen large random mutant libraries for variants of the enzymes with improved folding. However, generation of random mutant libraries often results in frequent generation of either frame shift mutations or stop codons. When screening for mutants with improved folding, it is therefore necessary to exclude the large number of clones that no longer express the target protein.
Current methods for analyzing protein expression and folding often focus on extraction of protein from the production organism, separating the protein into soluble (folded) and insoluble fractions, and analyzing these fractions using SDS-PAGE, dot blot based technologies, or by fusion of the target proteins to markers (Shih et al. 2002, Vincentelli et al. 2005, Wang et al. 2014). These are often time-consuming processes that are not amenable to screening of larger libraries of production organisms or protein variants at the single cell level. Other methods require the addition of large protein tags that may affect protein folding. For this reason, there is a need for a high throughput method that enables screening for protein folding and also protein translation at the single cell level.
Such a method would require either direct selection of bacterial growth based on for example antibiotics resistance or the possibility of sorting the production organisms based on the expression of for example a fluorescent marker.